1. Field of the Invention
The present invention is related to the fields of vaccinology, immunology and medicine. The invention provides compositions and methods for enhancing immunological responses against MelanA peptide analogues which are coupled, fused or attached otherwise to virus-like particles (VLPs) by binding, preferably by packaging immunostimulatory substances, in particular immunostimulatory nucleic acids, and even more particular oligonucleotides containing at least one non-methylated CpG sequence, into the VLPs. The invention can be used to induce strong and sustained T cell responses particularly useful for the treatment of tumors.
2. Related Art
The essence of the immune system is built on two separate foundation pillars: one is specific or adaptive immunity which is characterized by relatively slow response-kinetics and the ability to remember; the other is non-specific or innate immunity exhibiting rapid response-kinetics but lacking memory.
It is well established that the administration of purified proteins alone is usually not sufficient to elicit a strong immune response; isolated antigen generally must be given together with helper substances called adjuvants. Within these adjuvants, the administered antigen is protected against rapid degradation, and the adjuvant provides an extended release of a low level of antigen.
Unlike isolated proteins, viruses induce prompt and efficient immune responses in the absence of any adjuvants both with and without T-cell help (Bachmann & Zinkernagel, Ann. Rev. Immunol. 15:235-270 (1997)). Many viruses exhibit a quasi-crystalline surface that displays a regular array of epitopes which efficiently crosslinks epitope-specific immunoglobulins on B cells (Bachmann & Zinkernagel, Immunol. Today 17:553-558 (1996)). Viral structure is even linked to the generation of anti-antibodies in autoimmune disease and as a part of the natural response to pathogens (see Fehr, T., et al., J. Exp. Med. 185:1785-1792 (1997)). Thus, antigens on viral particles that are organized in an ordered and repetitive array are highly immunogenic since they can directly activate B cells and induce the generation of a cytotoxic T cell response, another crucial arm of the immune system.
Viral particles as antigens exhibit two advantages over their isolated components: (1) due to their highly repetitive surface structure, they are able to directly activate B cells, leading to high antibody titers and long-lasting B cell memory; and (2) viral particles, but not soluble proteins, have the potential to induce a cytotoxic T cell response, even if the viruses are non-infectious and adjuvants are absent.
In addition, DNA rich in non-methylated CG motifs (CpG), as present in bacteria and most non-vertebrates, exhibits a potent stimulatory activity on B cells, dendritic cells and other APC's in vitro as well as in vivo. Although bacterial DNA is immunostimulatory across many vertebrate species, the individual CpG motifs may differ. In fact, CpG motifs that stimulate mouse immune cells may not necessarily stimulate human immune cells and vice versa. In addition, immunostimulatory CpG-oligodeoxynucleotides induce strong side effects by causing extramedullary hemopoiesis accompanied by splenomegaly and lymphadenopathy in mice (Sparwasser et al., J. Immunol. (1999), 162:2368-74 and Example 18).
There have been remarkable advances made in vaccination strategies recently, yet there remains a need for improvement on existing strategies. In particular, there remains a need in the art for the development of new and improved vaccines that promote a strong CTL immune response and anti-pathogenic protection as efficiently as natural pathogens in the absence of generalized activation of APCs and other cells.
Melanomas are aggressive, frequently metastatic tumors derived from either melanocytes or melanocyte related nevus cells. Melanomas make up approximately three percent of all skin cancers and the worldwide increase in melanoma is unsurpassed by any other neoplasm with the exception of lung cancer in women. Even when melanoma is apparently localized to the skin, up to 30% of the patients will develop systemic metastasis and the majority will die. In the past decade immunotherapy and gene therapy have emerged as new and promising methods for treating melanoma, for example, treatment of Melanoma patients with the Melan A/MART-1 peptide with or without adjuvants. These Strategies are usually of limited success. Moreover, most of the studies did not measure directly the ex vivo CTL response with MHC class I multimers but rather used CTL cultures and stimulated them for several weeks before they could eventually measure a MelanA specific CTL response. In general, peptides are not immunogenic by itself and have a very short half life.
Another way of immunotherapy is the loading of dendritic cells with either the MelanA/MART-1 Peptide, or transfection of dendritic cells with MelanA/MART-1-RNA and re-injection onto patients. Drawback of this procedure is the purification and incubation of autologous dendritic cells from each individual patient for several days with cytokines in vitro. This is very delicate because the dendritic cells have to be in the right state of maturation for being immunogenic and not tolerogenic that could lead to T cells no responding to the tumor anymore.
Another approach from Dudley, M. E. (Science. 2002 Oct 25;298(5594):850-4) the is isolation of MelanA-specific T cells from autologous tumor-material of patients, in vitro cultivation and expansion and reinjection into the donor. As the aforementioned approach, a specific vaccine needs to produced separately for each individual patient and is therefore not the most efficient therapy.
Characterization of potent melanoma vaccines is, therefore, important for the development of new strategies for cancer immunotherapy, in particular for melanoma.